Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer Int. J. Cancer (Pred. Oncol.): 74, 45–49 (1997) r 1997 Wiley-Liss, Inc. DELETION OF THREE DISTINCT REGIONS ON CHROMOSOME 13q IN HUMAN NON-SMALL-CELL LUNG CANCER Kenji TAMURA,1 Xue ZHANG,1 Yoshinori MURAKAMI,1 Setsuo HIROHASHI,2 Hong-Ji XU,3 Shi-Xue HU4, William F. BENEDICT4 and Takao SEKIYA1* 1Oncogene Division, National Cancer Center Research Institute, Tokyo, Japan 2Pathology Division, National Cancer Center Research Institute, Tokyo, Japan 3M.D. Anderson Cancer Center, The Woodlands, TX, USA We examined loss of heterozygosity (LOH) at the retinoblastoma susceptibility gene (RB1) locus on chromosome 13q14 in 20 non-small-cell lung cancers (NSCLCs) using polymorphic markers. The expression of RB protein was examined by immunohistochemical analysis of paraffinembedded specimens of the same tumors. The results revealed that 10 of 16 informative cases showed an LOH at the RB1 locus, whereas only 2 of the 10 tumors lost expression of the RB protein. These 2 tumors had mutations in the remaining RB1 allele. Thus, inactivation of the RB1 gene appears to be involved in a small subset of NSCLCs only. To elucidate the presence of tumor-suppressor genes other than RB1 on 13q, heterozygosity at 15 different loci was investigated. Of 20 tumors analyzed, 15 showed an LOH at least at one locus, and the regions 13q12.1-qter, 13q12.2-14.2 and 13q14.1-q14.3, including the RB1 locus, were deleted in significant numbers of the tumors. Our results suggest that, in addition to the RB1 gene, abnormalities of other tumorsuppressor genes on chromosome 13q are involved in the development of human NSCLCs. Int. J. Cancer 74:45–49. r 1997 Wiley-Liss, Inc. The retinoblastoma susceptibility gene (RB1), which is located on the long arm of chromosome 13 (q14), is the first tumorsuppressor gene identified in human cancers (Friend et al., 1986; Fung et al., 1987; Lee et al., 1987). RB1 gene inactivation was revealed not only in retinoblastoma but also in many other human tumors, including osteosarcomas; soft tissue sarcomas; small-cell lung carcinomas; and breast, bladder, prostate, liver and esophageal cancers (see review: Benedict et al., 1990). In non-small-cell lung cancers (NSCLCs), Xu et al. (1991) and Reissman et al. (1993) have shown the absence of RB protein by immunohistochemical means in about one-third of tumors. We previously identified an LOH at the RB1 locus in 6 of 8 informative squamous-cell lung cancers. However, PCR-SSCP analysis of the remaining RB1 gene revealed inactivating mutations in only 2 of the 6 tumors (Sachse et al., 1994). The discrepancy between LOH at the RB1 locus and the loss of RB protein expression has also been reported on larger series of NSCLCs. Reissmann et al. (1993) demonstrated that only 8 of 25 tumors with loss of expression of the RB protein showed LOH at the RB1 locus, while one of 12 tumors with normal RB protein expression showed LOH at the RB1 locus. Gouyer et al. (1994) also demonstrated LOH at the RB1 locus in 11 NSCLC tumors, only 4 of which showed absence of the RB protein. In this study, we examined the allelic states of different loci on chromosome 13q together with the RB1 locus and the RB protein status in human NSCLCs. Our results indicated that inactivation of the RB1 gene was involved in only a subset of NSCLCs and that the presence of commonly deleted regions carried putative tumorsuppressor genes other than RB1. MATERIAL AND METHODS DNA samples Cancerous and non-cancerous tissues of the lung were resected surgically or obtained at autopsy from 20 patients with NSCLC at the National Cancer Hospital (Tokyo, Japan). High m.w. DNA was prepared from these specimens by the proteinase K-phenolchloroform extraction method. These cancers included 9 adenocarcinomas, 6 squamous-cell carcinomas, 2 adenosquamous carcinomas, 1 adenoid cystic carcinoma and 2 large-cell carcinomas. LOH analysis with polymorphic markers Nineteen polymorphic markers, 4 at the RB1 locus and 15 at other loci on chromosome 13q, were amplified by PCR for LOH analysis. Sixteen markers detected polymorphisms in microsatellite repeating sequences, while 2 and 1 detected those of single-base substitutions and variable (T)s, respectively. These features are summarized in Table I. Primers with reported nucleotide sequences (Murakami et al., 1991; Sachse et al., 1994; Zhang et al., 1994) were supplied by Bio-Synthesis (Lewisville, TX) and used to amplify DNA markers. The PCR mixture of 5 µl contained 50 ng of template DNA and primers labeled by the polynucleotide kinase reaction with [g-32P]ATP, as described (Orita et al., 1989b). Thirty cycles of the reaction proceeded at 94°C for 20 sec and at 56–60°C for 2 min in a Gene Amp PCR system 9600 (Perkin-Elmer Cetus, Emeryville, CA). The reaction mixture was diluted 10-fold with 95% formamide, 20 mM EDTA, 0.05% xylene cyanol and 0.05% bromophenol blue, then heated at 80–90°C for 5 min. To analyze DNA markers with microsatellite sequences and variable Ts, 1 µl of the diluted mixture was loaded onto a 5% polyacrylamide gel containing 7 M urea and resolved by electrophoresis at 50°C for 70–130 min. To detect a single-base substitution polymorphism by SSCP analysis (Orita et al., 1989a,b), the diluted mixture (1 µl) was subjected to electrophoresis in a non-denaturing 5% polyacrylamide gel containing 5% glycerol at 10°C for 200 min. These gels were dried on filter paper and exposed to Kodak XAR-5 film (Eastman-Kodak, Rochester, NY) at room temperature for 5–8 hr. Immunohistochemical staining RB nuclear protein was stained in paraffin-embedded sections as described (Xu et al., 1991), using the highly specific, affinitypurified, polyclonal anti-RB antibody RB-WL-1 (Zhang et al., 1994). Contract grant sponsor: Ministry of Health and Welfare of Japan grant-in-aid for the 2nd Comprehensive 10-Year Strategy for Cancer Control; Contract grant sponsor: Ministry of Health and Welfare of Japan grant for research on aging and Health; Contract grant sponsor: NIH National Cancer Institute, contract grant number CA54672. Xue Zhang’s present address is Department of Medical Genetics, China Medical University, Shenyang 110001, People’s Republic of China. *Correspondence to: Takao Sekiya, Oncogene Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104, Japan. Fax: 81-3-5565-9535. Received 28 June 1996; Revised 2 October 1996 TAMURA ET AL. 46 TABLE I – POLYMORPHIC MARKERS ON THE LONG ARM OF CHROMOSOME 13 3 3 Locus symbol Location Polymorphism Size range (bp) D13S141 4 D13S120 D13S139 D13S127 D13S126 D13S118 RB 1 (RB 1.20) RB 1 (RB intron 11) RB 1 (RB intron 21) RB 1 (RB intron 25) D13S227 D13S228 4 D13S133 D13S137 D13S119 D13S146 D13S131 D13S121 13q11 13q11–12.1 13q12.1 13q12.3–14.3 13q12.2–14.1 13q14.1–14.2 13q14.1–14.2 13q14.3 13q14.3 13q14.3 13q14.3 13q14.3 13q14.3 13q14.3 13q14.3 13q14.3–22 13q21.1–22 13q21.1–22 13q31 (CA)n (CA)n (CA)n (CA)n (CA)n (CA)n (CA)n (CTTT)n C/T Tn T/A (CA)n (CA)n (CA)n (CA)n (CA)n (CA)n (CA)n (CA)n 100–109 125–127 112–136 129–137 130–142 100–112 187–201 550–600 245 417 202 76–85 130–134 134–185 117–127 124–140 103–134 166–172 160–178 D13S175 1 1Brackets indicate that the order of the loci has not been determined. RESULTS Status of the RB1 gene and RB protein in NSCLCs The allelic state of RB1 was analyzed in 20 human NSCLCs using 4 polymorphic markers within the gene. As a representative result, detection of microsatellite sequence polymorphism and LOH by RB1.20 is shown in Figure 1. Similar analyses revealed LOH in the RB1 gene in 10 of 16 informative cases, as summarized in Table II. To establish whether the tumors with LOH at the RB1 locus also had mutations in the remaining RB1 allele, expression of the RB protein in tumors was examined immunohistochemically using paraffin-embedded specimens of the same set of tumors and polyclonal antibody against RB protein RB-WL-1. Representative results are shown in Figure 2. The nuclei of the cancer cells without loss of the RB1 allele as well as those of normal parenchymal cells of patient 141 were heterogeneously stained with the antibody (Fig. 2a). However, cancer cells with an allelic loss of the RB1 gene from patient 167 were not stained with the antibody, indicating inactivation of the remaining RB gene (Fig. 2b). In the patient, noncancerous cells surrounding the cancer cells were clearly stained. The results of the immunohistochemical analysis are summarized in Table II. All of the tumors that retained heterozygosity at the RB1 locus expressed RB protein. However, of 10 tumors with an LOH at the RB1 locus, only 2 that were known to have the mutated RB1 gene did not express RB protein. Analysis of LOH on chromosome 13q DNA from the same 20 patients with NSCLCs was examined for an LOH on 13q at various loci using 15 markers for polymorphic sequences together with polymorphic markers within the RB1 gene. Representative results of the LOH analysis are shown in Figure 3. A comparison of cancerous and normal DNA from patient 206 revealed an LOH at the D13S175 locus in DNA from tumors, while the DNA retained heterozygosity at the D13S120 locus. In tumor LuC137, there were LOHs at the RB1 and D13S228 loci but heterozygosity at the D13S133 locus was retained. Tumor LuC169 showed LOH at the D13S139 locus but not at either the D13S120 or the RB1 locus. Our results are summarized in Figure 4. Fifteen of 20 tumors had an LOH at least at one 13q locus analyzed. Five of them showed an LOH at all of the loci examined, suggesting loss of the entire chromosome 13. The results of a partial loss of chromosome 13q in the other 10 tumors indicated the presence of 3 separate regions commonly deleted in significant numbers of tumors. Regions I, II and III were from the centromeric region of 13q to the D13S120 locus (13q12.1), between the D13S139 (13q12.3–14.3) and D13S118 FIGURE 1 – Allelic losses at the RB1 locus in human NSCLCs. Polymorphic RB1.20 sequence was amplified by PCR from pairs of DNA from cancerous and non-cancerous tissues of patients. Heterozygosity at the RB1 locus was retained in the tumor in patient 169, while LOH was observed in the tumor in patient 137. N and C represent DNA from non-cancerous and cancerous tissues, respectively. TABLE II – ALLELLIC LOSS AT THE RB LOCUS, LOSS OF RB PROTEIN Tumor Histological type1 Stage Allelic loss2 RB protein Inactivating mutation5 LuC59 LuC104 LuC118 LuC128 LuC137 LuC141 LuC148 LuC150 LuC153 LuC161 LuC164 LuC166 LuC167 LuC168 LuC169 LuC190 LuC195 LuC200 LuC202 LuC206 Ad Adenosq La La Adenoid cyst Sq Sq Sq Ad Sq Ad Adenosq Sq Ad Ad Sq Ad Ad Ad Ad IV IV I I IIIa IIIa I IIIa IV I IIIa II I I I IIIa I IV IIIa IIIa 1 NI3 2 ND 1 1 1 1 NI 1 2 1 1 1 2 1 2 2 2 2 ND4 1 1 1 1 1 1 2 1 1 1 1 2 ND 1 1 1 1 1 1 2 26 2 2 2 2 2 17 2 2 2 2 18 2 2 2 2 2 26 2 1Ad, adenocarcinoma; Adenosq, adenosquamous-cell carcinoma; La, large-cell carcinoma; Adenoid cyst, adenoid cystic carcinoma; Sq, squamous-cell carcinoma.—21 and 2 indicate allelic loss and retention of 2 alleles, respectively.—3NI, not informative.–4ND, not done.– 5Mutations were detected previously (Sachse et al., 1994).—6LuC104 and LuC202 carried cancer-specific mutations with unknown function, CAC to CCC (codon 673, exon 20) and G to T (intron 14), respectively.–7Abnormal splicing; GTAAG to GCAAG (intron 17).— 8Frameshift: AAG to AG (codon 96, exon 3). DELETION OF 13q IN HUMAN NSCLC 47 FIGURE 2 – Immunohistochemical staining of RB nuclear protein. (above) An RB-positive tumor, LuC 141, showed an LOH at the RB1 locus but no mutations in the remaining allele. (below) An RB-negative tumor, LuC 167, showed an LOH at the RB1 locus and a mutation in the remaining allele. RB protein expression was detected by staining with the antibody RB-WL-1. Scale bar: 10 µm. (13q14.1–14.2) loci and between the D13S118 (13q14.1–14.2) and D13S133 (13q14.3) loci, respectively. Region III includes the RB1 locus. DISCUSSION Tumor tissues in which DNAs were analyzed for LOH might have contained normal cells. In contrast, immunohistochemical analysis of paraffin-embedded sections of these specimens was straightforward and would detect expression of the RB protein directly in each of the cancer cells. Therefore, the results obtained for RB protein were unambiguous. Thus, immunohistochemical analysis has been used as a sensitive method for detection of RB1 gene inactivation, though the results of the analyses are known to be dependent on the quality of the antibodies tested (Xu et al., 1991; Reissmann et al., 1993; Geradts et al., 1996). In the present study, we examined expression of the RB protein with anti-RB antibody, RB-WL-1, and confirmed the discrepancy between LOH at the RB1 locus and loss of expression of the RB protein. This antibody has been widely used to detect RB protein expression in various tumors (Xu et al., 1991, 1993, 1994; Zhang et al., 1994). In this study, we detected 2 tumors, LuC150 and LuC167, lacking RB protein expression. Only these tumors showed an LOH 48 TAMURA ET AL. FIGURE 3 – Allelic losses at loci on chromosome 13q in human NSCLCs. Polymorphic sequences were amplified by PCR from pairs of DNA samples from cancerous and non-cancerous tissues of patients. Products were resolved by denaturing polyacrylamide gel electrophoresis. N and C represent DNA from non-cancerous and cancerous sources, respectively. at the RB1 locus as well as an inactivating mutation in the remaining RB1 allele (Table II). The coincident results indicate that antibody RB-WL-1 is highly specific to RB protein. In contrast, RB protein was detected in the other tumors with an LOH at the RB1 locus. These results indicated that inactivation of the RB1 gene was certainly involved but in only a subset of NSCLCs. We identified 3 commonly deleted regions (I–III) by extensive analyses of LOH on chromosome 13q. LOHs could be produced by either interstitial deletion or translocation of the chromosome. It may be possible that chromosome 13 contains unstable regions where chromosomal breakage could occur with a high frequency. As shown in Figure 4, region I was suggested by LOHs in tumors LuC150, LuC202 and LuC206; region II by those in LuC164, LuC169 and LuC202; and region III by those in LuC137, LuC153 and LuC166. Regions I and II, which might carry tumor-suppressor genes other than RB1, were located closer to the centromere, while region III included the RB1 locus. However, in the 3 tumors carrying LOH at region III, RB protein was expressed. Therefore, the presence of a tumor-suppressor gene other than RB1 was suggested. Frequent LOH on chromosome 13q and less frequent RB1 mutations have also been found in various human tumors, including breast cancers (Borg et al., 1992), head and neck cancers (Yoo et al., 1994) and pituitary tumors (Pei et al., 1995). Detailed FIGURE 4 – Allelic losses at the loci on the long arm of chromosome 13 in NSCLCs. Patient numbers are indicated at the top of the panel. Loci analyzed are indicated at the left side of the panel. Loci in the box have not yet been placed in order. d and s, Loss and retention of heterozygosity, respectively. Vertical lines indicate loci where the signals were not informative. Solid bars on the right indicate commonly deleted regions. analyses of head and neck cancers and pituitary tumors have indicated that commonly deleted regions also include the RB1 locus and overlap region III identified in the present study. Schutte et al. (1995) have reported that a restricted fragment within region II at 13q12.3 is homozygously deleted in human pancreatic cancers. In this region, the BRCA2 gene for susceptibility to hereditary breast cancer has been identified (Wooster et al., 1995). It has been reported that the BRCA2 gene and the RB1 gene could be distinct target loci for allelic imbalance at chromosome 13 in sporadic breast cancer (Hammann et al., 1996). The relationship between tumor-suppressor genes on 13q in NSCLCs and those in other tumors remains to be clarified. 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